Numerous detection and analysis methods for determining physiological parameters in body fluid samples are based on photometric measuring principles. Photometric methods allow the qualitative and quantitative detection of analytes in liquid samples.
In many cases, clinically relevant parameters, examples including the concentration or the activity of an analyte, are determined by mixing an aliquot of a body fluid from a patient with one or more assay reagents in vitro, initiating a biochemical reaction which brings about a measurable change in an optical property of the assay preparation. Photometry examines and utilizes the attenuation of a luminous flux upon penetration through an absorbing and/or scattering medium. Different photometric measurement methods allowing the measurement of a turbid liquid assay preparation are used depending on the nature of the triggered biochemical or biophysical reaction.
To this end, it is possible to use turbidimetric methods, in which the turbidity or the optical density of a solution or suspension is measured on the basis of the light attenuation or absorbance of a light beam passing directly through the suspension.
The intensity of the light beam decreases upon penetration through a measurement cell or cuvette containing a liquid sample. The losses can be influenced by interactions of the light beam with the sample situated in the measurement cell, for example by absorption, diffraction, scattering and/or reflection effects. It is generally possible for diffraction, scattering and reflection effects to be disregarded or compensated for by reference measurements, so that mainly absorption contributes to the attenuation of the light beam.
Photometric determinations of concentration are therefore based on a law where the absorbance or absorption is dependent on the concentration of the dissolved substances and the layer thickness of the measurement cell at a certain wavelength of the incident light. This relationship is described by the Beer-Lambert law:E(λ)=−log(I/I0)=ε(λ)·c·d where E(λ) is the absorbance dependent on the wavelength λ of the light beam, I is the light intensity after penetration through the sample, I0 is the light intensity before penetration through the sample, ε(λ) is the wavelength-dependent molar extinction coefficient of a transirradiated substance, c is the molar concentration of the transirradiated substance, and d is the layer thickness transirradiated by the light beam, for example of the measurement cell.
On the basis of the absorbance E(λ) of a sample, it is possible to ascertain the concentration of a substance in a solution. To this end, it is necessary for the absorbance of at least one standard solution of known concentration to have been determined beforehand. Since absorbance is proportional to concentration, it is possible to ascertain the concentration of a dissolved substance in an unknown sample by means of calibration by absorbance measurements of multiple standard solutions of known concentrations.
However, the absorbance of a sample depends not only on the concentration of the substance to be determined itself, but also on the nature of the sample matrix. The absorbances of various substances are additive in a mixture, if the substances do not interact with one another. Body fluids, examples including blood plasma or blood serum, are complex mixtures and contain not only the analyte to be determined, but also a multiplicity of further substances which influence the total absorption of the sample.
However, in individual cases, body fluid samples may contain abnormally high concentrations of one or more intrinsic, i.e., endogenous, substances which turn out to be interfering in photometric detection methods when a tolerable concentration is exceeded and may have an effect in relation to a systematic error.
It is known that problems are caused by hemolytic, icteric and/or lipemic serum or plasma samples, which have abnormally high hemoglobin, bilirubin and/or lipid concentrations. Abnormally high concentrations of these interfering substances may be caused by a pathological state of the patient or else by an improper acquisition or storage of sample. If such samples are subjected to a photometric method used for determining an analytical, diagnostically relevant parameter, there is the risk of an incorrect determination, the result of which may be possibly a misdiagnosis and, in the worst case, an incorrect treatment of the patient. The preanalytical identification of hemolytic, icteric and lipemic samples is thus particularly important for avoiding faulty analysis results.
Therefore, there is a need for methods for ascertaining the spectrometric effects of interfering substances in body fluid samples or for identifying body fluid samples containing elevated concentrations of one or more interfering substances.
EP-A1-1059522, U.S. Pat. No. 4,263,512, US 2009/0009750 A1 and US 2010/0174491 A1 describe various methods for determining bilirubin, hemoglobin and lipids in plasma or serum samples. For example, in EP-A1-1059522, the absorbance which remains after subtraction of the absorbance due to hemoglobin and bilirubin and which contains in particular the absorbance caused by lipids is subjected to local linear approximation.
However, even the last-mentioned method has the disadvantage that specifically a comparatively high lipid concentration may influence the determination of bilirubin and hemoglobin in the same sample and thus distort the measurement values.
WO 2013/010970 A1 already describes a method which allows, even in the presence of high lipid concentrations, an accurate determination of bilirubin and hemoglobin and also a determination of lipid concentration in one sample. The method described in WO 2013/010970 A1 fundamentally comprises the measurement of the absorbance of the sample at various wavelengths, the calculation of a power-function approximation curve for the absorbance of the lipids, the subtraction of the hemoglobin and bilirubin share of the absorbances until a lipid curve remains, and lastly—for determining the lipid content—the division of a thus theoretically ascertained lipid absorbance value by the extinction coefficient specific for lipids.
With the known method, it has been observed that it is possible to correctly determine the lipid content in only approximately 20% of all samples. In the case of the remaining samples, the determined lipid content deviates from the true lipid content by more than +/−25%.